Identification and characterization of new factors in the framework of Arabidopsis thaliana miRNA function

Abstract

One of the major forces steering differentiation and fate specification is transcription. A main regulator controlling transcript accumulation are small RNAs (sRNAs). Within those, microRNAs (miRNAs) are amidst the most abundant components, and important factors in regulation of protein coding gene expression in higher eukaryotes. Their biogenesis and processing are well characterized in animals, and a large number of protein complexes and cofactors involved have been identified and studied. In plants, considerably fewer details are known, and especially the regulation of miRNA biogenesis and function is far from understood. In recent years, numerous screens were employed to enlighten this labyrinth. Within the frame of my thesis, I characterized three novel cofactors involved in miRNA biogenesis and function that were identified by two forward genetics screens established in our laboratory, and broadened our knowledge about additional regulatory layers. The first screen yielded two candidates characterized here, TRANSCRIPTIONAL DEFECTS OF HPR1 BY OVEREXPRESSION 2 (THO2) and the REGULATOR OF CBF GENE EXPRESSION 3 (RCF3). We found that THO2 is associated with miRNA precursors and their processing, as well as with splicing. This substantiates the connection between miRNA pathways, transcription and splicing, and adds detail to the picture of co-transcriptional miRNA precursor processing. RCF3, on the other hand, proved to influence miRNA levels and action prevalently in young, dividing tissues, presumably via affecting the phosphorylation and thus activity of a major miRNA processing factor. These findings shift the focus to tissue-biased action as novel regulatory layer and emphasize the importance of chemical modifications to modulate miRNA co-factors’ function. The third candidate studied in my thesis, HAWAIIAN SKIRT (HWS), stems from a screen looking for modifiers of miR156 function. I genetically and molecularly connect HWS to the general miRNA framework. Function of HWS in this setting appears to be dependent on the activity of its F-box domain, presumably in the context of an Skp-Cullin-F-box (SCF) complex that normally targets substrates for ubiquitination.